Adenosine A 2A receptor ligand recognition and signaling is blocked by A 2B receptors.

The adenosine receptor (AR) subtypes A2A and A2B are rhodopsin-like Gs protein-coupled receptors whose expression is highly regulated under pathological, e.g. hypoxic, ischemic and inflammatory conditions. Both receptors play important roles in inflammatory and neurodegenerative diseases, are blocked by caffeine, and have now become major drug targets in immuno-oncology. By Förster resonance energy transfer (FRET), bioluminescence resonance energy transfer (BRET), bimolecular fluorescence complementation (BiFC) and proximity ligation assays (PLA) we demonstrated A2A -A2B AR heteromeric complex formation. Moreover we observed a dramatically altered pharmacology of the A2A AR when co-expressed with the A2B AR (A2B ≥ A2A ) in recombinant as well as in native cells. In the presence of A2B ARs, A2A -selective ligands lost high affinity binding to A2A ARs and displayed strongly reduced potency in cAMP accumulation and dynamic mass redistribution (DMR) assays. These results have major implications for the use of A2A AR ligands as drugs as they will fail to modulate the receptor in an A2A -A2B heteromer context. Accordingly, A2A -A2B AR heteromers represent novel pharmacological targets.